This in vitro study investigated the metabolism of human osteoarthritic (OA) chondrocytes encapsulated in a spherical matrix enriched of chitosan. Human OA chondrocytes were encapsulated and cultured for ... [more ▼]

This in vitro study investigated the metabolism of human osteoarthritic (OA) chondrocytes encapsulated in a spherical matrix enriched of chitosan. Human OA chondrocytes were encapsulated and cultured for 28 days either in chitosan-alginate beads or in alginate beads. The beads were formed by slowly passed dropwise either the chitosan 0.6%- alginate 1.2% or the alginate 1.2% solution through a syringe into a 102 mM CaCl2 solution. Beads were analyzed histologically after 28 days. Interleukin (IL)-6 and -8, prostaglandin (PG) E2, matrix metalloproteinases (MMPs), hyaluronan and aggrecan were quantified directly in the culture supernatant by specific ELISA and nitric oxide (NO) by using a colorimetric method based on the Griess reaction. Hematoxylin and eosin staining showed that chitosan was homogeneously distributed through the matrix and was in direct contact with chondrocytes. The production of IL-6, IL-8 and MMP-3 by chondrocytes significantly decreased in chitosan-alginate beads compared to alginate beads. PGE2 and NO decreased also significantly but only during the first three days of culture. Hyaluronan and aggrecan production tended to increase in chitosan-alginate beads after 28 days of culture. Chitosan-alginate beads reduced the production of inflammatory and catabolic mediators by OA chondrocytes and tended to stimulate the synthesis of cartilage matrix components. These particular effects indicate that chitosan-alginate beads are an interesting scaffold for chondrocytes encapsulation before transplantation to repair cartilage defects. [less ▲]

Objective: The aim of this study was to compare the gene expression pattern of synovial cells from inflammatory (I) or normal/reactive (N/R) areas of a synovial membrane harvested from the same ... [more ▼]

Objective: The aim of this study was to compare the gene expression pattern of synovial cells from inflammatory (I) or normal/reactive (N/R) areas of a synovial membrane harvested from the same osteoarthritis (OA) patient. Methods: Synovial tissues were obtained from 12 knee OA patients at the time of total knee replacement. The inflammatory status of the synovial membrane was characterized according to macroscopic criteria and sorted as N/R and I. Biopsies were cultured separately for 7 days. Microarray gene expression profiling between N/R and I areas was performed. Western blot and immunohistochemistry confirmed the identified genes that were differentially expressed. Results: 896 differentially expressed genes between N/R and I zones were identified. The key pathways were related to inflammation, cartilage metabolism, Wnt signaling and angiogenesis. In the inflammatory network, TREM1 and S100A9 were strongly up-regulated. MMP-3 and -9, cathepsin H and S were significantly up-regulated in the cartilage catabolism pathway, whereas the most up-regulated anabolism enzyme was HAS1. Wnt-5A and LRP5 were up-regulated whereas FZD2 and DKK3 were down-regulated in the Wnt signaling. Finally, STC1, a protein involved in angiogenesis was identified as the most up-regulated gene in I zones compared to N/R zones. Conclusion: This study is the first to identify different expression pattern between two areas of the synovial membrane in the same patient. These differences concern several key pathways involved in OA pathogenesis. This analysis also provides information regarding new genes and proteins as potential targets for the future therapeutic. (c) 2013 American College of Rheumatology. [less ▲]

Objective: This study aimed to evaluate the structural benefit of a new biomaterial composed of alginate-chitosan (AC) beads dispersed in an hydrogel (H) derived from chitosan on the development of ... [more ▼]

Objective: This study aimed to evaluate the structural benefit of a new biomaterial composed of alginate-chitosan (AC) beads dispersed in an hydrogel (H) derived from chitosan on the development of osteoarthritis (OA) in rabbit. Design: OA was induced by the surgical transection of the anterior cruciate ligament in rabbits. Animals received a single intra-articular injection (900 μl) of AC beads in H hydrogel, H hydrogel alone or saline one week after surgery. OA development was followed by X-rays. Blood samples were collected throughout the study to measure biological markers (PGE2 and CRP). Macroscopic observation and histological evaluation of articular cartilage and synovial membrane were performed 6 weeks after surgery. Results: AC beads in H hydrogel prevented from the development of OA based on the reduction of the Kellgren & Lawrence (K&L) score. It also significantly reduced the histological score of cartilage lesion severity. This effect was homogenous on every joint compartment. It was due to a significant effect on cartilage structure and cellularity scores. The injection of AC beads in H hydrogel also tended to reduce the synovial membrane inflammation. No significant variation of biological markers was noted. Conclusions: The present pilot study provides interesting and promising results for the use of AC beads in H hydrogel in animal. It indeed prevented the development of OA cartilage lesions without inflammatory signs. The potencies of this biomaterial to protect OA joint should be further documented. It could then represent a new alternative for viscosupplementation in human OA management. [less ▲]

in Osteoarthritis and Cartilage (2013, April), 21(Supplement April 2013),

Purpose: Chondroitin sulfate (CS) is one the most used molecules in the management of OA. In this study, we performed a microarray analysis and identified a differential expression profile between control ... [more ▼]

Purpose: Chondroitin sulfate (CS) is one the most used molecules in the management of OA. In this study, we performed a microarray analysis and identified a differential expression profile between control and IL-1β stimulated synovial fibroblast cells cultures. In a second step, we investigated the effects of CS on this gene expression profile. Methods: OA synovial specimens were obtained from 12 patients undergoing knee replacement. At the surgery time, the synovial membrane was dissected. Synovial fibroblast cells (SFC) were enzymatically isolated and used after four passages (P4). SFC were pre-treated 1 hour with highly purified bovine CS (200 µg/ml, Bioibérica S.A., Barcelona, Spain) before treatment with IL-1β (1 ng/ml) for 24 hours. Total RNA was extracted using the RNeasy Mini Kit. RNA purity and quality were evaluated using the Experion RNA StdSens Analysis kit (Bio-rad Laboratories). Gene expression profiling was performed using Illumina’s multi-sample format Human HT-12 BeadChip (Illumina Inc.). Differential analysis was performed with the BRB array tools software. Class comparison test between control (Ctl) and interleukin (IL)-1β conditions, Ctl and Ctl/CS and IL-1β and IL-1β/CS conditions was based on paired t-test where Ctl and IL-1β, Ctl and Ctl/CS and IL-1β and IL-1β/CS were paired for each patient. The biological relevance of up- and down-regulated genes was analyses with Ingenuity Pathways Analysis (Ingenuity® Systems). Probes with a p-value below 0.001 were chosen and classified as up- or down-regulated ones. Results: 3308 genes were identified as differentially expressed genes between Ctl and IL-1β conditions. We observed a differential profile of expression of major pathways involved in OA pathogenesis. The key identified pathways were related to inflammation, complement cascade, angiogenesis, cartilage catabolism and anabolism and Wnt signaling. In the inflammatory network, the most upregulated cytokines were IL-8 and IL-6 with a fold change of 156.25 and 58.8 respectively. We also identified several chemokines, enzymes and metallothioneins (MTs). Complement factor B (CFB) and complement component 3 (C3) are two factors upregulated in the inflammatory complement cascade. We also identified some genes implicated in the angiogenesis pathway. The most upregulated was Stanniocalcin 1 (STC1) with a fold change of 9.09. The differential expression of intermediates involved in both cartilage anabolism and catabolism was revealed by the IL-1β stimulation, showing an imbalance in favour of catabolism. MMP-3 was largely upregulated (fold change of 62.5). Wnt 5A and low density lipoprotein receptor-related protein (LRP8) were significantly upregulated while frizzled homolog 2 (FZD2) and dickkopf homolog 3 (DKK3) were downregulated in the Wnt signaling pathway. We next performed a class comparison test between Ctl and Ctl/CS in one hand and IL-1β and IL-1β/CS on the other hand. 660 genes were identified as differentially expressed between Ctl and Ctl/CS conditions while 241 genes were identified between IL-1β and IL-1β/CS. Among them, our attention was focused on two genes upregulated in the presence of CS: lysyl oxidase-like 4 (LOXL4) and claudin 11 (CDLN11), two genes that negatively regulate cell invasion. Conclusions: We here evidenced in synovial fibroblast cells the modulation of gene expression following IL-1β stimulation. We also demonstrated the modulatory effects of CS on gene expression and isolated several CS-modulated genes of interest such as LOXL4 and CDLN11, which could constitute new mechanisms of action of the molecule and contribute to explain the symptomatic efficacy of CS in the treatment of OA. [less ▲]

in Osteoarthritis and Cartilage (2013, April), 21(Supplement April 2013), 69

Purpose To evaluate the effects of a single intra-articular injection of a new biomaterial consisting in a mix of alginate-chitosan (AC) beads and a viscous thermogelling chitosan-based (H) hydrogel on ... [more ▼]

Purpose To evaluate the effects of a single intra-articular injection of a new biomaterial consisting in a mix of alginate-chitosan (AC) beads and a viscous thermogelling chitosan-based (H) hydrogel on cartilage lesion in osteoarthritis (OA) rabbit model. These effects were compared to those obtained with the intra-articular injection of either chitosan-based (H) hydrogel without the AC bead or saline solution. Methods OA was surgically induced by the transection of the anterior cruciate ligament (ACLT) in HYLA albino rabbits. One week after surgery, animals were randomly divided into 3 groups: group I (n=7): mix of AC beads and H hydrogel; group II (n=7): H hydrogel alone; group III (n=7): saline solution (control). The treatments (900 µl) were injected intra-articularly. X-rays from the right knee were performed before surgery, at the time of injection and at sacrifice. The standard radiographs were acquired in extension and scored by the Kellgren and Lawrence (K&L) scale. After 6 weeks, animals were euthanized and the right joint was dissected. The macroscopic evaluation of cartilage from femoral condyles and tibial plateaus stained with India ink was done. Histological sections stained with Safranine-O/fast green from bearing areas of each compartment were evaluated according to the OARSI histological score. Briefly, the evaluation considered: staining of the cartilage matrix (0-6), cartilage structure (0-11), chondrocyte density (0-4) and cluster formation (0-3), where 0 represented a normal situation and 24 points the maximum severity score. Blood samples were collected the day of injection and prior the sacrifice. Prostaglandin E2 (PGE2) and C-reactive protein (CRP) were measured in serum using immunoassays. Results The X-rays analysis showed a significant decrease (p <0.05) of the K&L score in group I (AC beads and H hydrogel; 1.5 ± 0.2) compared with group II (H hydrogel; 2.2 ± 0.5) and group III (saline solution; 3.0 ± 0.4). The size and the severity of the macroscopic OA cartilage lesion tended to decrease in group I compared to the other groups. The histological global score that refers to all compartments of the knee joint was significantly decreased in group I (11.0 ± 0.7) compared to group II (14.4 ± 0.6, p <0.01) and group III (14.8 ± 0.6, p <0.001). No significant variation of PGE2 and CRP serum levels were observed in each after 6 weeks follow-up whatever the treatment injected. Conclusions This study showed that a biphasic hydrogel composed by AC beads and H hydrogel prevented OA in rabbit with ACL transection. This effect was not observed with the hydrogel alone, suggesting that AC beads play a role in joint protection. The preventive effect was observed in all joint compartments indicating a global protective effect of this new viscosupplementation. [less ▲]

Introduction Osteoarthritis (OA) is the most prevalent arthritic disease. It is characterized by the degradation of articular cartilage accompanied by the inflammation of the synovial membrane and ... [more ▼]

Introduction Osteoarthritis (OA) is the most prevalent arthritic disease. It is characterized by the degradation of articular cartilage accompanied by the inflammation of the synovial membrane and sclerosis of subchondral bone. OA produces pain and loss of joint function. Today, there is no treatment to cure OA or to delay effectively its progression. Current treatments are mainly based on alleviation of painful symptoms but are unable to restore the cartilage. The development of new scaffold for tissue engineering is a promising approach. Herein, we report the effects of alginate-chitosan hydrogel (AC) beads on the metabolism of chondrocytes. Materials and Methods Human chondrocytes were isolated from OA cartilage and cultured either in AC beads or in alginate (A) beads. AC beads were prepared using chitosan (KiOmedine-CsU ultra-pure chitosan from KitoZyme, Herstal, Belgium) and alginate. The two polymer solutions were prepared separately before being mixed together. Cells were added to the polymer mixture and the cell-containing beads prepared by precipitation in a calcium chloride solution. The chondrocytes embedded in the beads were then cultured in a well defined culture medium for up to 28 days. Cell viability was determined by quantifying the release of lactate deshydrogenase (LDH) in the culture supernatant. Interleukin (IL)-6 and -8, prostaglandin E2 (PGE2), matrix metalloprotease (MMP)-3 and aggrecan were measured by specific ELISA. Finally, nitric oxide (NO) was measured by the Griess reaction. Results Histological analysis of AC beads showed chondrocytes in contact with chitosan trabeculae that were homogeneously distributed in the alginate matrix. LDH level remained below the limit of detection over the culture duration suggesting that AC had no cytotoxic effect. By comparison with culture in A beads, chondrocytes in AC beads produced significantly higher amounts of aggrecan but lowered the levels of MMP-3, NO, IL-6, IL-8 and PGE2. Discussion The contact between cells and AC beads components led us to hypothesize that chitosan has beneficial effects such as anti-inflammatory, anti-catabolic and stimulating effects on cartilage matrix components. Conclusion These particular effects indicate that AC beads are potentially new carriers for cell transplantation, particularly to repair cartilage defects. They could be further developed under various formulations, such as microbeads in combination with hydrogel for efficient viscossuplementation. [less ▲]